I. Field of the Invention
The present invention is directed to an agent and method for eliminating ascorbate interference in reagent systems, particularly assay systems using oxidase/peroxidase coupled reactions or similar type redox chemistry. The present invention can also be used in reagent systems involving enzyme/substrate reactions in which the substrate is sensitive to reductants such as ascorbate. Thus, the present invention can be used to remove interference of ascorbate in analyte detection systems for the determination of glucose, occult blood, cholesterol, triglycerides and uric acid, as well as other analytes. More specifically, the agent and method of this invention comprise the use of water insoluble cerium (IV) compounds.
II. Discussion of the Prior Art
Ascorbic acid is an important and well known nutrient which exists naturally in many foods, such as fruits and vegetables, and which can also be synthesized inexpensively as a food additive or vitamin supplement. As a result, ascorbic acid is relatively plentiful and the general population tends to ingest more ascorbic acid than necessary.
Excess ascorbic acid is generally not harmful because the body will only absorb ascorbic acid in an amount sufficient to meet the body's short term needs, quickly disposing of the excess by means of the body's urinary system. As a result, ascorbic acid is often found in urine samples used in medical analysis.
Unfortunately, ascorbic acid in urine can be an unwanted interferant for many urine assays presently in existence. Urine assays in general are important medical tools in diagnosing and treating the general population and therefore much attention has been focused upon this problem in recent years.
Urine assays often comprise redox indicator and these indicators are generally incorporated into the assay system in their reduced form. The indicators will change color when oxidized and are therefore called "redox" indicators, because they change color as they move from a reduced to an oxidized state due to the presence of an oxidizer.
In many assay systems an analyte of interest will directly or indirectly cause the oxidation of the assay's redox indicator, thereby causing a detectable response which correlates with the presence of the analyte. In other words, when the appropriate analyte is added to the assay system the redox indicators will undergo the following reaction: EQU I.sub.red .fwdarw.I.sub.ox +at least 1 electron
wherein I.sub.red is the indicator in a reduced state (negative color response) and wherein I.sub.ox is the indicator in an oxidized state (positive color response).
However, ascorbic acid in solution undergoes the following reaction: ##STR1## where A is defined as C.sub.4 H.sub.6 O.sub.4. Ascorbate is a reductant, because it is able to donate an electron and thereby reduce the substance receiving the electron: ##STR2## In this way, ascorbate can interfere with a redox indicator by reducing the indicator when it is in its oxidized form, thereby inhibiting the intended color change and causing a "false negative."
Numerous methods have been tried, some successfully, which eliminate the adverse affect ascorbic acid can have upon redox indicators in an assay system. One successful method is disclosed in U.S. Pat. No. 4,587,220 by Mayambala-Mwanika, et al., whereby ferric complexes are combined with hydroperoxide to act as an ascorbate scavenger. This scavenger system prevents ascorbate reduction of redox indicators.
In Mayambala, Fe.sup.+3 is complexed with an iron chelate, such as HEDTA (N-2-hydroxyethylethylene-diaminetriacetic acid), and reacted with ascorbic acid. The Fe(III) chelate scavenges ascorbic acid as follows: ##STR3##
The Mayambala scavenger causes the secondary alcohol groups in ascorbic acid to be oxidized into ketones and in this way ascorbic acid is transformed into a nonreductant compound which will not interfere with a redox indicator system. However, the Fe(II)-HEDTA is recycled back into Fe(III)-HEDTA using a peroxide or hydroperoxide as follows: ##STR4## The resulting hydroxide radical can receive an electron according to the following reaction: EQU HO.spsp..+1 electron.fwdarw.HO.sup.-
and can therefore act as an oxidizer. As a result, the hydroxide radical can oxidize the redox indicator when the indicator is in its reduced (original) form, thereby causing a color change regardless of whether or not an analyte of interest is present. Consequently, in preventing a false negative due to ascorbic acid, the ascorbic acid scavenger system of Mayambala can cause an unwanted false positive. In solving one problem, the ascorbic acid scavenger system creates another.
The Mayambala patent suggests an adjustment of the assay's pH by means of conventional buffers to minimize the oxidation affect of the scavenger system. Indeed, since oxidation reactions are typically acid catalyzed and different oxidation reactions can involve different kinetic interactions it may be possible to find a pH which would accelerate indicator oxidation due to the analyte without substantially accelerating indicator oxidation due to the ascorbic acid scavenger system.
The Mayambala patent also suggests a dry phase format which separates the reagent system into two dip solutions. One dip solution is incorporated and dried on a support and thereafter the second dip solution is also incorporated and dried onto the support. Mayambala therefore suggests that certain constituents of a reagent system can perhaps be separated in a dry format in a manner where the desired oxidation reaction will dominate over the unwanted oxidation reaction.
However, pH adjustment may not prove to be advantageous for every reagent system and a multidip process for creating a dry phase system may not always be practical or workable for every reagent system. Furthermore, a liquid format may in some cases be more desirable than a dry phase format.
Consequently, it is an object of the present invention to provide a system whereby ascorbate or similar type reductant interference can be eliminated without adversely affecting the reliability of a redox indicator system.
A further object of the present invention is to provide an ascorbate resistant assay system which can be used in solution form or can be incorporated into a reagent strip using a one dip method.
Other objects and features of the present invention will become apparent to one of ordinary skill in the art upon the reading of the following specification, particularly the detailed description of the preferred embodiments and the claims.